Clin Exp Immunol 1999; 116:28 32
`
`Immunomodulation by vitamin B12: augmentation of CD8+ T lymphocytes and
`natural killer (NK) cell activity in vitamin B12-deficient patients by methyl-B12
`treatment
`
`J. TAMURA, K. KUBOTA*, H. MURAKAMI’~, M. SAWAMURA, T. MATSUSHIMA, T. TAMURA, T. SAITOH,
`H. KURABAYSHI* & T. NARUSE Third Department of Internal Medicine, Gunma University School of Medicine, Maebashi,
`*Department of Internal Medicine, Kusatsu Branch Hospital, Gunma University Hospital, Kusatsu, and ~ School of Health Sciences,
`Gunma UniversitZ. Maebashi Japan
`
`(Accepted for publication 7 January 1999)
`
`SUMMARY
`
`It has been suggested that vitamin B 12 (vit.B 12) plays an important role in immune system regulation,
`but the details are still obscure. In order to examine the action ofvit.B12 on cells of the human immune
`system, lymphocyte subpopulations and NK cell activity ~vere evaluated in 11 patients ~vith vit.B12
`deficiency anaemia and in 13 control subjects. Decreases in the number of lymphocytes and CD8+ cells
`and in the proportion of CD4+ cells, an abnormally high CD4/CD8 ratio, and suppressed NK cell
`activity ~vere noted in patients compared ~vith control subjects. In all 11 patients and eight control
`subjects, these immune parameters ~vere evaluated before and after methyl-B12 injection. The
`lymphocyte counts and number of CD8+ cells increased both in patients and in control subjects. The
`high CD4/CD8 ratio and suppressed NK cell activity ~vere improved by methyl-B12 treatment.
`Augmentation of CD3 CD16+ cells occurred in patients after methyl-B12 treatment. In contrast,
`antibody-dependent cell-mediated cytotoxicity (ADCC) activity, lectin-stimulated lymphocyte blast
`formation, and serum levels of immunoglobulins ~vere not changed by methyl-B12 treatment. These
`results indicate that vit.B 12 might play an important role in cellular immunity, especially relativing to
`CD8+ cells and the NK cell system, ~vhich sugge sts effects on cytotoxic cells. We conclude that vit.B 12
`acts as an immunomodulator for cellular immunity.
`
`Keywords vitamin B12 NK cell CD8 immunomodulation
`
`INTRODUCTION
`
`Vitamin B 12 (Vit.B 12) has various effects on biological processes
`in vivo. It is ~vell kno~vn that megaloblastic anaemia and peripheral
`nerve disturbances are caused by lack of vit.B12. In the immune
`system, an important role of vit.B12 has been reported. Vit.B12
`enhanced T cell proliferative responses to concanavalin A (Con A)
`and immunoglobulin synthesis of B cells by poke~veed mitogen
`(PWM) [1]. It has been reported that vit.B12 deficiency caused
`suppression of protective immune responses to viruses and bacteria
`in an animal model [2].
`In human immunity, the action of vit.B12 is still obscure,
`probably because it is impossible to study the action of vit.B12
`using artificially deficient human model systems. Ho~vever, ~ve
`occasionally encounter patients ~vith vit.B 12 deficiency disorders,
`
`such as megaloblastic anaemia, and it is possible to observe the
`change of immunological parameters after vit.B12 administration
`in such patients. A fe~v investigations and case reports of immu-
`nological abnormalities in vit.B12-deficient megaloblastic anae-
`mia patients have been reported [3 7], but there have been no
`systematic studies.
`In order to investigate the biological actions ofvit.B 12 on cells
`of the human immune system, lymphocyte subsets and NK cell
`activities ~vere examined in patients ~vith vit.B12 deficiency
`anaemia, and the changes of immune parameters after vit.B12
`administration ~vere evaluated.
`
`SUBJECTS AND METHODS
`
`Correspondence: J. Tamura, Third Department of Internal Medicine,
`Gunma University School of Medicine, 3 Showa-machi, Maebashi
`371-8511, Japan.
`
`Subjects
`Eleven ne~vly diagnosed Japanese patients ~vith vit.B 12 deficiency
`
`anaemia ~vere admitted to our hospital bet~veen December 1990
`
`28
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`Vit.B12 augments CD8+ cells and NK cell activity
`
`29
`
`and June 1993 (age 36 83 years, median 65 years; six males and five
`females). Seven of 11 patients had pernicious anaemia (PA) and four
`had post-gastrectomy megaloblastic anaemia (PGMA). All patients
`shelved lo~v serum levels of vit.B12 (< 85 pg/ml; normal range 230
`820pg/ml). Diagnosis ~vas made based on medical history,
`macrocytic anaemia in peripheral blood, erythroblastosis ~vith
`megaloblastic changes in bone marro~v, lo~v serum levels of
`vit.B12, the presence of anti-intrinsic factor antibody, antiparietal
`antibody and clinical responsiveness to vit.B12 therapy.
`Thirteen haematologically and immunologically normal volun-
`teers ~vere included as a control group (age 26 92years, median
`72years; five males, eight females). None shelved lo~v serum
`levels of vit.B12 or anaemia. All tests, including any sampling of
`blood, ~vere performed ~vith informed consent and ~vith our
`hospital ethical committee’s approval.
`
`also evaluated in four patients. In all patients and control subjects
`serum levels of IgG, IgA and IgM ~vere evaluated before and after
`methyl-B 12 administration.
`
`Statistical analysis
`Statistical analysis ~vere conducted ~vith paired t-test for the
`comparison ~vithin each group of patients and control subjects
`and ~vith unpaired t-test for the comparison bet~veen the t~vo groups
`as indicated in the legend for Table 1. Obtained P values ~vere
`re-estimated ~vith Wilcoxon signed rank test and Mann Whitney
`rank sum test. Significance ~vas defined as follo~vs: both t-test and
`non-parametric method shelved P< 0.05. P values < 0.05 obtained
`~vith t-test but not ~vith non-parametric analysis ~vere regarded as
`not significant but shelving a tendency. Analysed values ~vere
`represented as mean -- s.d.
`
`Study design
`Leucocyte and lymphocyte numbers, percentage and absolute
`numbers of CD4+ cells and CD8+ cells, CD4/CD8 ratio and NK
`cell activity ~vere evaluated in all patients at diagnosis and
`compared ~vith the values in control subjects.
`In order to examine the immunomodulatory effect of vit.B12,
`methylcobalamin ~vas administrated to all patients and to eight of
`13 volunteers as follo~vs. Methyl-vit.B12 (500~g/day; methyl-
`B 12; mecobalamin; Eisai, Tokyo, Japan) ~vas injected intramuscu-
`larly every other day for 2~veeks and immunophenotyping of
`peripheral lymphocytes and NK cell activity ~vere evaluated as
`before treatment. At that time, all patients and control subjects
`shelved high serum levels of vit.B 12 (> 3000 pg/ml). After 2 ~veeks
`treatment, patients ~vere treated ~vith vit.B121000~g every
`3 months as out-patients; all of them ~vere quite ~vell and anaemia
`had improved. After 1 2 years of follo~v up, NK cell activity ~vas
`estimated in seven of 11 patients ~vho shelved high serum levels of
`vit.B12 (> 3000 pg/ml).
`
`Surface marker analysis
`Heparinized peripheral blood ~vas obtained and the mononuclear
`cell (MNC) fraction ~vas collected by centrifugation on lympho-
`cyte separation medium (Gunma Immunology Institute, Fujioka,
`Japan). After ~vashing ~vith PBS, MNC ~vere used for further
`analysis. Commercial MoAbs used for immunophenotyping ~vere
`as follo~vs: CD3 (OKT3 FITC), CD4 (OKT4 FITC), CD8
`(OKT8 FITC) (Ortho, Raritan, N J) for both patient group and
`control group before and after methyl-B12 treatment, CD56
`(NKH1 FITC) (Coulter, Hialeah, FL) for nine of 11 patients
`before and after treatment. Additionally, t~vo-colour analyses,
`using CD3 (Leu-4 PE) xCD57 (Leu-7 FITC), CD3 (Leu-4 PE)
`xCD16 (Leu-lla FITC), CD57 (Leu-7 FITC) xCD16 (Leu-
`1 lc PE) (Becton Dickinson, Mountain Vie~v, CA), ~vere done in
`eight of 11 patients and 10 of 13 control subjects for evaluation of
`NK cell subsets. All phenotyping ~vas performed using a FACScan
`(Becton Dickinson).
`
`NK cell activity and other tests for immune response
`NK cell activity ~vas estimated in all cases and all control subjects
`before and after 2 ~veeks of methyl-B 12 treatment by the standard
`SlCr_release assay using K562 cells as target cells (effector:target
`ratio ~vas 20:1) and results ~vere expressed as percentage cell lysis.
`In five patients, phytohaemagglutinin (PHA)-, ConA- and
`PWM-stimulated lymphocyte blast formation ~vere measured,
`and antibody-dependent cell-mediated cytotoxicity (ADCC) ~vas
`
`RESULTS
`
`Immunophenotyping and NK cell activity in patients and control
`subjects before methyl B12 administration
`Although no significant difference in leucocyte counts ~vas noted
`bet~veen patients (n= 11) and control subjects (n= 13) (4100--
`1600//d versus 5363 -- 1367//d; NS), the lymphocyte counts ~vere
`significantly decreased in patients compared ~vith control subjects
`(1414-- 695/~1 versus 2110-- 669/~1; P< 0.01).
`The proportion of CD4+ cells ~vas also significantly elevated in
`patients (48.1 -- 10.5% versus 34.5 -- 8.7%; P<0.01); ho~vever,
`the absolute number of CD4+ cells ~vas not different from that in
`controls (711 -- 435//d versus 714 -- 357//d; NS). In contrast,
`~vhile the slight decrease in the proportion of CD8+ cells ~vas not
`significant (19.9 -- 7.0% versus 24.5 -- 9.6%; NS), the absolute
`number of CD8+ cells ~vas significantly smaller in patients than in
`control subjects (276 -- 148/~1 versus481 +_ 177/~1; P<0.01). The
`CD4/CD8 ratio ~vas significantly elevated in patients (3.0 -- 1.7
`versus 1.7 -- 0.8; P<0.05).
`Suppressed NK cell activity ~vas clearly seen in patients
`compared ~vith control subjects (12.9 -- 7.4% versus
`52.5 -- 14.8%; P<0.01).
`
`Effect of methyl B12 administration on lymphocyte subsets and NK
`cell activity in patients and control subjects
`As mentioned above, leucocyte counts and lymphocyte counts,
`CD4+, CD8+, CD56+ cell counts and NK cell activity ~vere
`measured at the end of the 2-~veek treatment ~vith methyl-B12.
`Results of statistical analysis of immunological parameters before
`and after methyl-B12 administration in both patients and control
`subjects are summarized in Table 1. The leucocyte counts and
`lymphocyte counts of patients ~vere increased significantly after
`methyl-B 12 treatment (P< 0.05). After treatment, the lymphocyte
`counts ~vas still significantly louver in patients than in control
`subjects (P< 0.05). Interestingly, an increase in the lymphocyte
`counts ~vas observed even in control subjects (P< 0.05).
`As sho~vn in Table 1, a significant decrease of percentage
`CD4+ cells ~vas observed in patients after treatment (P<0.01),
`~vhile no significant change ~vas noted in control subjects. No
`significant change of the absolute number of CD4+ cells ~vas
`observed in patients after methyl-B12 treatment, but a slight
`increase ~vas observed in control subjects (NS but tendency).
`An increase in percentage CD8+ cells after methyl-B12 treat-
`ment ~vas noted in patients (P<0.05), but not in control subjects.
`
`© 1999 Blackwell Science Ltd, Clinical and Experimental Immunology, 116:28 32
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`30
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`J. Tamura et al.
`
`Table 1. Change of immune p~xameters before and after 2 weeks of methyl-B 12 administration in patients and control subjects
`
`Patients (n- 11)
`
`Control subjects (n- 8)
`
`Before
`
`After
`
`Before
`
`After
`
`No. of leucocytes (/~l)
`No.of lymphocytes (/~1)
`Percent CD4+ cells
`
`No. ofCD4+ cells (/~1)
`Percent CD8+ cells
`
`No. ofCD8+ cells (/~1)
`CD4/CD8 ratio
`NK cell activity (%)
`
`4100 + 1600
`1414 + 69577
`48"1 + 10"577
`711 + 435
`19"1 + 7"0
`276 + 14877
`3"0 + 1 "7?
`12"9 + 7"4??
`
`5500 + 1800"
`1802 + 737*?
`41 "8 + 10"0"*
`757 + 378
`23"1 + 6"8*
`411 + 198"*?
`2"1 + 1 "1"
`28"9 + 15"3"*?
`
`5200 + 1188
`2213 + 491
`38"9 + 6"8
`870 + 305
`23"8 + 6"4
`489 + 129
`1 "7 + 0"8
`54"0 + 15"0
`
`5342 + 965
`2703 + 792*
`39"4 + 10"1
`1108 + 614(*)
`24"1 + 7"1
`596 + 191"
`1"7 + 0"8
`53"0 + 13"0
`
`The mean value + s.d. of 11 (patients) and eight (control subjects) is given.
`*,**Signaificant change of parameters after methyl-B12 injection within each group of patients or control subjects (P< 0"05 and P< 0"01, respectively).
`?,??Signaificant difference of parameters of patients before and after methyl-B12 treatment comp~xed with those of control subjects (P<0"05, P<0"01,
`respectively).
`(*)P< 0"05 was obtained with t-test but not with non-p~xametric analysis.
`
`Increases in the absolute number of CD8+ cells ~vere noted in both
`patients and control subjects (P<0.01, P<0.05, respectively);
`ho~vever, the absolute number of CD8+ cells in patients after
`treatment ~vas still louver than that in control subjects (P< 0.05).
`The CD4/CD8 ratio ~vas significantly decreased by methyl-B 12
`treatment in patients (P< 0.05), but not in control subjects, and the
`difference bet~veen patients and control subjects disappeared after
`methyl-B 12 administration.
`In patients, the decreased level of NK cell activity ~vas restored
`by methyl-B 12 administration (P< 0.01); ho~vever, the level of NK
`cell activity ~vas still louver than that of the control group
`(P< 0.05). In control subjects, NK cell activity ~vas not changed
`by methyl-B12 treatment. After 1 2years of follo~v up, ~vith
`methyl-B 12 administration (1000/~g injection for every
`3 months), further restoration of NK cell activity ~vas observed in
`patients compared ~vith that observed after 2 ~veeks of methyl-B 12
`treatment (40.3 -- 11.9% versus 28.9 -- 15.3%; P< 0.01; n = 7, 11,
`respectively) and the restored NK cell activity ~vas comparable to
`that of control subjects (40.3 -- 11.9% versus 53.0-- 13.0%; NS;
`n = 7, 8, respectively).
`
`Effects of methyl B12 treatment on NK cell subsets and other
`immunological parameters
`The percentage and absolute number of CD56+ cells ~vere esti-
`mated in nine patients before and after methyl-B 12 treatment, and
`compared ~vith those in 10 control subjects. Both proportion and
`absolute number of CD56+ cells in patients before methyl-B12
`administration ~vere louver than those in control subjects (13.9 --
`6.1% versus 23.7 -- 9.8%; P< 0.05; n = 9, 10, respectively; 191.5 --
`64.9//~1 versus 461.8--237.3//& P<0.01; n=9, 10, respectively).
`After methyl-B12 administration, the proportion of CD56+ cells
`~vas not changed (14.3 -- 5.8% versus 15.9 -- 6.3%; NS; n=9).
`Although the slight increase in absolute number of CD56+ cells
`after methyl-B12 treatment in patients ~vas not significant
`(191.5 -- 64.9//~1 versus 333.2 -- 209.1//& NS (P= 0.09); n = 9),
`the difference bet~veen patients and control subjects disappeared
`after methyl-B 12 administration.
`On the other hand, a slight increase in absolute number of
`CD3 CD16+ cells ~vas noted (146.7--70.4//~1 versus 237.0--
`
`120.4//~1; F< 0.05; n = 7); ho~vever, both values ~vere significantly
`(P<0.01) louver than that of normal subjects (477.2-- 193.3//~1).
`Similarly, absolute numbers of CD16+ CD57+ cells in patients
`before treatment ~vere significantly louver than that in control
`subjects (129.8 -- 55.0//~1 versus 367.9 -- 235.2//& F< 0.05; n = 7,
`10, respectively). After methyl-B12 treatment, a slight increase in
`absolute number of CD16+CD57+ cells in patients ~vas noted but
`~vas not significant (129.8 -- 55.0//~1 versus 201.5 -- 136.9//& NS;
`n= 7); ho~vever, the difference bet~veen patients and control
`subjects disappeared after methyl-B12 administration. No other
`parameters of double-staining analyses of CD3 xCD16, CD8 x
`CD57, CD 16 x CD57 ~vere significantly changed (data not sho~vn).
`PHA-, ConA-, and PWM-stimulated blast formation of
`lymphocytes ~vas measured in five patients before and after
`methyl-B 12 treatment, but no suppression or change ~vas observed
`after treatment (all results ~vere in the normal range, data not
`sho~vn). ADCC ~vas evaluated in three patients but no suppression
`of activity or other significant change ~vas observed before or after
`treatment ~vith vit.B12 (all results ~vere in the normal range, data
`not sho~vn). Serum levels of immunoglobulins in patients and
`control subjects ~vere in the normal range and no change ~vas
`observed after methyl-B 12 treatment (data not sho~vn).
`
`Follow up of patients
`In all patients, anaemia ~vas improved ~vithin 2 4 ~veeks and the
`patients remained ~vell thereafter. No adverse effects ~vere seen in
`patients or control subjects treated ~vith methyl-B12.
`
`DISCUSSION
`
`In the present study ~ve have demonstrated various immunomodu-
`
`latory effects of vit.B12. Serum levels of immunoglobulins ~vere
`
`not affected by vit.B 12 deficiency or supplementation. A decrease
`in the absolute numbers of lymphocytes, especially CD8+ cells,
`
`and an increase in the CD4/CD8 ratio in vit.B 12-deficient patients
`
`~vere found. Vit.B12 treatment led to an increase in the number of
`lymphocytes, including CD8+ cells, not only in patients but also in
`
`control subjects, and to a significant increase of NK cell activity in
`patients.
`
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`
`Vit.B12 augments CD8+ cells and NK cell activity
`
`31
`
`These results are consistent ~vith others referred to above [3 5]
`
`In clinical studies of immunological or neurological disorders
`
`and ~vith clinical observations that ~ve reported previously [6,7]. In
`
`such as autoimmune diseases and HIV infections, some effects of
`
`contrast, Soler ot aL [8] and Carmel ot ~1. [9] found no significant
`decrease in CD8+ cells nor a significantly increased CD4/CD8
`
`vit.B12 have been reported. Sandyk et al. reported a relationship
`
`bet~veen vit.B12 and onset of multiple sclerosis (MS) [17]. They
`
`ratio in PA. Although differences of the races of the subjects can
`
`discussed the possibility of involvement of vit.B12 as a cause of
`
`not be ignored, as Carmel ot ~1. discussed [9], the design of the
`
`MS through effects on immune system regulation. A patient ~vith
`
`study is also likely to be an important factor explaining the
`
`AIDS dementia complex ~vas apparently successfully treated
`
`differences. In our study, only ne~vly diagnosed, untreated patients
`
`~vith vit.B12 [18], and a relationship bet~veen vit.B12 deficiency
`
`~vere included, and methyl-B12 ~vas administered to all patients
`
`and the development of immune dysfunction in AIDS has been
`
`using the same procedure and the same drug.
`
`reported [19].
`
`In order to examine the effects of vit.B12, methyl-B12 ~vas
`
`used, because mecobalamin is kno~vn to be one of the strongest
`
`In conclusion, ~ve found a significant decrease in the absolute
`number of CD8+ cells and suppressed NK cell activity in vit.B 12-
`
`immunomodulators among the vit.B12 derivatives [1]. Methyl-
`
`deficient patients. These abnormalities could be at least partly
`
`B 12 restored the abnormally high CD4/CD8 ratio and significantly
`
`restored by methyl-B12 treatment. Moreover, augmentation of
`
`decreased the number of CD8+ cells. In contrast, the absolute
`number of CD4+ cells ~vas not significantly changed by methyl-
`
`CD8+ cells by methyl-B12 treatment ~vas observed even in control
`
`subjects. These observations may contribute to our understanding
`
`B12 in patients. Our observations suggest that a decrease in the
`
`of the potential anti-tumour effects of vit.B12, and may partly
`
`absolute number of CD8+ cells is the main abnormality of the
`
`explain the high risk of gastric carcinoma in PA; our data also
`
`lymphocyte phenotype ~vhich could be restored by methyl-B12.
`
`provide a rationale for considering the use ofvit.B 12 for treating a
`
`Although a basic investigation of the biochemical effects of
`
`variety of other immunological, neurological, and oncological
`
`vit.B12 on DNA synthesis has been reported [10], details of the
`
`disorders.
`
`mechanism of immunomodulation by vit.B12 are still unkno~vn.
`
`Ho~vever, studies on the effects of vit.B12 on cell apoptosis have
`
`been reported [11,12], and ~ve speculated that the prevention of
`
`apoptosis of lymphocytes by vit.B 12 might be one explanation for
`
`immunological abnormalities in megaloblastic anaemia. Decreases
`in the absolute numbers of lymphocytes and CD8+ cells ~vere
`
`observed in our patients and these ~vere partly restored by vit.B 12
`
`administration. These reports and the present observations suggest
`that CD8÷ cells are one of the most sensitive subpopulations to
`
`apoptosis caused by lack of vit.B12. Ho~vever, this is only
`
`speculation and further investigation is needed.
`
`In addition, suppressed NK cell activity in vit.B12-deficient
`patients and partial restoration of NK cell activity by 2 ~veeks of
`
`vit.B12 treatment ~vere observed in the present study. After 1
`
`2 years of treatment, complete restoration ~vas observed in patients
`
`~vho sho~ved high serum levels of vit.B12. It is not clear ~vhether
`
`the incomplete restoration of NK cell activity is due to too short a
`
`period of treatment to prevent apoptosis. Long-term follo~v up of
`
`patients should be useful for further understanding this problem.
`+
`Augmentation of the CD3 CD16 fraction and CD16+CD57+
`
`fraction, ~vhich possess strong NK cell activity [13], suggesting
`
`anti-tumour activity, ~vas observed after vit.B12 therapy. A high
`
`risk of malignancies, especially gastric carcinoma, in PA has been
`
`demonstrated [14]. While Rode et al. [15] suggests that the
`
`increased risk of gastric tumours is due to the proliferation of
`
`endocrine cells induced by hypergastrinaemia in PA, a deficiency
`of vit.B12 causing lo~v numbers of CD8+ lymphocytes and
`
`depressed NK cell activity may be additional risk factors.
`
`On the other hand, the possibility of an anti-tumour effect of
`
`methyl-B12 ~vas reported using an experimental model of cancer
`
`[16]. In that report, enhancement of PHA-, ConA- and PWM-
`
`stimulated lymphocyte blast formation by methyl-B12 ~vas sug-
`
`gested to be one of the mechanisms of anti-tumour immunity. In
`
`our study, PHA-, ConA- and PWM-stimulated lymphocyte blast
`formation and ADCC ~vere measured in some patients, but no
`
`suppression or change after methyl-B12 treatment ~vas noted.
`
`Although these negative results might be due to the small sample
`
`size in this study, it is possible that lymphocyte blast formation and
`
`ADCC do not play an important role in anti-tumour immunity
`compared ~vith the effects of CD8+ and NK cell activity.
`
`ACKNOWLEDGMENTS
`
`This study was supported in part by groats for research frorn the Ministry of
`Education, Science and Culture of Japan. We thank Dr Jun Tsuchiya, Dr
`Takuo Shirakura, Dr Toyoho Morita, Dr Toshirnasa Arai, Dr Kirnio Morita,
`Dr Hitushi Katahira, Dr Hitushi T~J~e, and Dr Masahiko Shinoh~xa for their
`help with this study.
`
`REFERENCES
`
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`2 Vellerna P, Rutten VP, Hock A ot al. The effect of cobalt supplernenta-
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`4 Wodzinski MA, Forrest MJ, Barnett D ot al. Lyrnphocyte subpopula-
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`7 Kubota K, Kurabayashi H, Kawada E ot al. Restoration of abnornaally
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`8 Soler J, Rernacha A, Nietu M, Ginaferrer E. Lyrnphocyte subpopula-
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`9 Carnael R, Boone D, Parker JW. Lyrnphocyte surface phenotypes in
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`10 Pfohl-Leszkowicz A, Keith G, Dirheinaer G. Effect of cobalarnin
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`can act as a rnethyl donor. Biochern 1991; 30:8045 51.
`11 Bunting RW, Seling MK, Dickersin GR. Apoptotic cells in peripheral
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`Pathol 1997; 29:223 7.
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`
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`JOINT 1031-0004
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`

`
`32
`
`J. Tamura et al.
`
`12 Ingrain CF, Davidoff AN, Marais E et al. Evaluation of DNA maalysis
`for evidence of apoptosis in megaloblastic anaemia. Br J Haematol
`1997; 96:576 83.
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